Muscle gene expression is a marker of amyotrophic lateral sclerosis severity.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset degenerative disease characterized by the loss of upper and lower motor neurons leading to progressive muscle atrophy and paralysis. The lack of molecular markers of the progression of disease is detrimental to clinical practice and therapeutic trials. OBJECTIVE: This study was designed to identify gene expression changes in skeletal muscle that could reliably define the degree of disease severity. METHODS: Gene expression profiles were obtained from the deltoid muscles of ALS patients and healthy subjects. Changes in differentially expressed genes were compared to the status of deltoid muscle disability, as determined by manual muscle testing, electrophysiology and the degree of myofiber atrophy. Functionally related genes were grouped by annotation analysis, and deltoid muscle injury was predicted using binary tree classifiers. RESULTS: Two sets of 25 and 70 transcripts appeared differentially regulated exclusively in early and advanced states of deltoid muscle impairment, respectively. The expression of another set of 198 transcripts correlated with a composite score of muscle injury combining manual muscle testing and histological examination. From the totality of these expression changes, 155 transcripts distinguished advanced from early deltoid muscle impairment with 80% sensitivity and 100% specificity. Nine of these transcripts, known also to be regulated in ALS mouse and surgically denervated muscle, predicted the advanced disease status with 100% sensitivity and specificity. CONCLUSION: We provide robust gene expression changes that can be of practical use when monitoring ALS status and the effects of disease-modifying drugs.

Gene profiling of skeletal muscle in an amyotrophic lateral sclerosis mouse model.

Muscle atrophy is a major hallmark of amyotrophic lateral sclerosis (ALS), the most frequent adult-onset motor neuron disease. To define the full set of alterations in gene expression in skeletal muscle during the course of the disease, we used the G86R superoxide dismutase-1 transgenic mouse model of ALS and performed high-density oligonucleotide microarrays. We compared these data to those obtained by axotomy-induced denervation. A major set of gene regulations in G86R muscles resembled those of surgically denervated muscles, but many others appeared specific to the ALS condition. The first significant transcriptional changes appeared in a subpopulation of mice before the onset of overt clinical symptoms and motor neuron death. These early changes affected genes involved in detoxification (e.g., ALDH3, metallothionein-2, and thioredoxin-1) and regeneration (e.g., BTG1, RB1, and RUNX1) but also tissue degradation (e.g., C/EBPdelta and DDIT4) and cell death (e.g., ankyrin repeat domain-1, CDKN1A, GADD45alpha, and PEG3). Of particular interest, metallothionein-1 and -2, ATF3, cathepsin-Z, and galectin-3 genes appeared, among others, commonly regulated in both skeletal muscle (our present data) and spinal motor neurons (as previously reported) of paralyzed ALS mice. The importance of these findings is twofold. First, they designate the distal part of the motor unit as a primary site of disease. Second, they identify specific gene regulations to be explored in the search for therapeutic strategies that could alleviate disease before motor neuron death manifests clinically.

Tissue specificity and regulation of the N-terminal diversity of reticulon 3.

Over the last few years, the widely distributed family of reticulons (RTNs) is receiving renewed interest because of the implication of RTN4/Nogo in neurite regeneration. Four genes were identified in mammals and are referred to as RTN1, 2, 3 and the neurite outgrowth inhibitor RTN4/Nogo. In the present paper, we describe the existence of five new isoforms of RTN3 that differ in their N-termini, and analysed their tissue distribution and expression in neurons. We redefined the structure of human and murine rtn3 genes, and identified two supplementary exons that may generate up to seven putative isoforms arising by alternative splicing or differential promoter usage. We confirmed the presence of five of these isoforms at the mRNA and protein levels, and showed their preferential expression in the central nervous system. We analysed rtn3 expression in the cerebellum further, and observed increased levels of several of the RTN3 isoforms during cerebellum development and during in vitro maturation of cerebellar granule cells. This pattern of expression paralleled that shown by RTN4/Nogo isoforms. Specifically, RTN3A1 expression was down-regulated upon cell death of cerebellar granule neurons triggered by potassium deprivation. Altogether, our results demonstrate that the rtn3 gene generates multiple isoforms varying in their N-termini, and that their expression is tightly regulated in neurons. These findings suggest that RTN3 isoforms may contribute, by as yet unknown mechanisms, to neuronal survival and plasticity.

Up-regulation of mitochondrial uncoupling protein 3 reveals an early muscular metabolic defect in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder affecting primarily motor neurons. Growing evidence suggests a mitochondrial defect in ALS. The precise molecular mechanisms underlying those defects are unknown. We studied the expression of mitochondrial uncoupling proteins (UCPs), key regulators of mitochondrial functions, in tissues from a mouse model of ALS (SOD1 G86R transgenic mice) and from muscular biopsies of human sporadic ALS. Surprisingly, in SOD1 G86R mice, UCPs, and particularly UCP3, were upregulated in skeletal muscle but not in spinal cord. Consistent with this pattern of expression, ATP levels were selectively depleted in muscle but not in neural tissues 1 month before disease onset and the respiratory control ratio of isolated mitochondria is decreased. UCP3 up-regulation was not observed in experimentally denervated muscles, suggesting that changes in muscular UCP3 expression are associated with the physiopathological processes of ALS. This is further supported by our observation of increased UCP3 levels in human ALS muscular biopsies. We propose that UCP3 up-regulation in skeletal muscle contributes to the characteristic mitochondrial damage of ALS and to the onset of the disease. Moreover, since skeletal muscle is a key metabolic tissue, our findings suggest that ALS may not solely arise from neuronal events but also from more generalized metabolic defects.

Early activation of antioxidant mechanisms in muscle of mutant Cu/Zn-superoxide dismutase-linked amyotrophic lateral sclerosis mice.

A subset of familial ALS cases is associated with missense mutations in the gene encoding Cu/Zn-superoxide dismutase (SOD1), a free radical scavenging enzyme that protects cells against oxidative stress. Overexpression of these ALS-linked mutations confers an unidentified gain of function to the enzyme that triggers a series of neurological disorders characteristic of human ALS. To understand how skeletal muscle may counteract the progression of the disease, we explored the expression of different molecular effectors involved in antioxidant pathways. Our results are strongly indicative of the early and long-lasting activation of a series of molecular effectors thought to act coordinately in preventing the increased oxidative stress characteristic of ALS.

Nogo provides a molecular marker for diagnosis of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by the selective degeneration of upper and lower motor neurons. The lack of a molecular diagnostic marker is of increasing concern in view of the therapeutic strategies in development. Using an unbiased subtractive suppressive hybridization screen we have identified a clone encoding the neurite outgrowth inhibitor Nogo and shown that its isoforms display a characteristic altered expression in ALS. This was first confirmed by analyzing Nogo isoform expression in a transgenic ALS model at early asymptomatic stages where we found increased levels of Nogo-A and decreased Nogo-C and importantly, not following experimentally induced denervation. Furthermore, we confirmed these changes in both post-mortem and biopsy samples from diagnosed ALS patients but not control patients. Thus, the alteration in Nogo expression pattern, common to sporadic and familial ALS, represents a potential diagnosis tool and points strongly to Nogo having a central role in disease.